Targeting oncogenic RNAs with antibodies Non-coding RNAs (ncRNAs) have been recognized as important cancer disease markers and therapeutic targets. The ncRNAs often form extensive structures and exert their oncogenic functions by interacting with protein partners, making the RNA-protein interfaces important targets for cancer therapy. However, currently there is a lack of a robust and general approach in targeting structured RNAs. The long-term goal is to develop antibody-based therapeutic drugs targeting structured oncogenic RNAs at the RNA-protein interfaces. Based on the PI's pioneer work in anti-RNA antibody phage selection, the central hypothesis is that through well-designed phage displayed libraries, Fabs can be engineered to bind RNA structure specifically and block the key RNA-protein interactions important in cancer cell proliferation and tumorigenesis. In the present proposal, the short-term goal is to develop potent Fab (antigen binding fragments) libraries for structured RNA recognition and use phage display to select specific Fabs binding to a model oncogenic ncRNA, initiator tRNA, for potential cancer pathway mediation. Recently, overexpression of human initiator tRNA (fully conserved in all vertebrates) was shown to drive cell proliferation and cause tumor formation in mice, suggesting initiator tRNA as a potential cancer drug target. The research objective will be achieved by pursuing the following specific aims. Aim 1, to develop effective ways to select RNA-binding antibodies, key parameters will be identified for Fab-RNA recognition and applied to design libraries tailored specifically for anti- RNA Fab selection. Based on preliminary data and literature, the working hypothesis is that systematic design of the variable sites and amino acid composition in library randomization will improve the efficacy of anti-RNA Fab libraries. Aim 2, as a proof-of-concept, specific Fabs will be obtained and characterized for their ability to bind oncogenic initiator tRNA and mediate cancer-related biological processes. In the cell-free assay, the specific Fabs will be tested for their interference in human in vitro translation. After engineered into bispecific antibodies with anti-HER2 Fab as the delivery arm, these antibodies will be delivered to HER2 positive cancer cell lines to test their anti-cancer effect. Completion of this work will not only generate anti- initiator tRNA Fabs for future cancer drug design but also provide a robust and general therapeutic approach to target extensively structured oncogenic RNAs, which is a challenge for antisense approaches. In addition, the bispecific antibody approach employs an innovative way to achieve high efficiency and specificity in cancer cell delivery that targets intracellular antigens.